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Identification of Gram Negative Bacteria in the Laboratory

written by: Teresa Martin•edited by: dianahardin•updated: 4/18/2011

Gram negative bacteria identification involves a series of tests in which the bacteria reacts with chemicals. Positive or negative reactions define the type of bacteria present and provide information regarding the best type of antibiotic to use for treatment.

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    Gram Stain and Culture for Gram Negative Bacteria

    The first step in gram negative bacteria identification involves doing a gram stain and culture. Gram negative bacteria grow well on MacConkey agar, which provides substances to inhibit the growth of gram positive bacteria. This isolates bacteria which are gram negative. Once the colonies have had the chance to grow for 18-24 hours, further testing will be performed to determine which type of gram negative bacteria is present in a specimen.

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    Carbohydrate Fermentation Test

    Some gram negative bacteria contain beta-galactosidase, an enzyme which acts upon lactose, breaking it down into glucose and galactose. Gram negative bacteria identification through the carbohydrate fermentation test will be positive if the enzyme beta-galactosidase is present. Usually, a broth containing lactose also contains phenol red, which turns yellow under acidic conditions. The conversion of lactose to glucose and galactose produces acidic conditions. Escherichia coli is an example of a lactose positive gram negative bacteria.

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    SIM Test

    The SIM test is actually three tests in one. The "S" stands for hydrogen sulfide. The principle of this test is to see if the organism can metabolize sulfur and produce hydrogen sulfide. A positive test for hydrogen sulfide will appear black in the agar.

    The "I" stands for Indole. Tryptophan, an amino acid, contains an amine group and indole. If the bacteria can break this amino acid into an amine group and indole, then the test is positive.

    The "M" stands for motility. Some bacteria are mobile in the media and some are not. The agar is inoculated by stabbing it. If growth only appears in the area of the stab, then the test is negative. If, however, growth appears throughout the medium, then it is positive for motility.

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    Citrate Test

    The citrate test is used in gram negative bacteria identification. Bacteria with citrate permease can alter agar containing citric acid, resulting in an alkaline pH. The agar turns from green to blue, if positive, and remains green if negative. Examples of gram negative bacteria which are citrate positive include Citrobacter freundii, which usually does not cause an infection. In certain circumstances, this bacteria can cause a urinary tract infection.

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    Phenylalanine Deaminase Test

    Bacteria containing phenylalanine deaminase, when inoculated on phenylalanine agar, can cause a transformation by removing the amino acid. The result is phenolpyruvic acid. When ferric acid is added to the agar slant, a positive result is demonstrated by a greenish color. Proteus species are examples of gram negative bacteria which are positive for the phenylalanine deaminase test.

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    Urease Test

    Bacteria containing the enzyme urease can breakdown urea into urea into ammonia and carbon dioxide. Ammonium hydroxide is formed with the addition of water and turns the broth a purple color.

    The positive or negative test results of each of the above mentioned tests provide physicians with a means for determining the properties of most gram negative bacteria and identifying the ones which may be causing an infection. There are other special tests for identifying less common gram negative bacteria.